The process of aerobic fermentation provides an important mechanism for the formation of certain products, such as enzymes, antibiotics, diagnostics and therapeutics. Such products are typically produced by the overexpression of a protein in cells of a microorganism, such as E. coli or S. cerevisiae. 
On a laboratory scale, such substances of interest are typically produced by preparing a “starter” culture which is then used to inoculate a larger volume of culture medium. Typically, the culture medium is contained within a conical flask and is placed in an incubator at a desired temperature. The growth of cells in the culture medium is monitored by intermittent sampling of small amounts of the culture and measurements of the optical density of the culture medium with the use of an external spectrophotometer. By way of explanation, measuring the optical density at approximately 600 nm detects scattering of light by microorganisms and the resulting OD reading is proportional to the cell density. The cells grow and divide and, typically, when the optical density of the culture has reached a desired value, the overexpression of a protein of interest is induced, for example, by a change in temperature or the addition of an inducer, etc.
Aerobic fermentation is routinely carried out on an industrial scale in stirred tanks. Typically, an optical probe is used to measure the optical density of the culture medium according to the Beer Lambert Law, which is defined by the following equation:OD=E.L. log10(Io/I)wherein                E=Extinction coefficient;        L=Path length of cell;        Io=Incident light intensity;        I=transmitted light intensity        
In industrial-scale fermentations, vigorous agitation and aeration is normally required in order to support optimal growth of the microorganisms or cells because they need to respire. Under such conditions, as much as a fifth of the overall aerated liquid volume of the culture medium may comprise gas bubbles dispersed in the liquid. Good liquid mixing of aerated medium means that many of these gas bubbles are small enough to pass through the light path of the optical probe used to measure the cell density of the culture. The presence of gas bubbles in the culture medium may have an effect on the apparent cell density of the medium, because gas bubbles have different light-scattering properties compared to the culture medium and the cells that are suspended therein. Scattering of light by bubbles in the medium tends to cause an overestimation of the apparent optical density of the medium. In particular, at low cell densities (where the gas bubble to cell ratio is greatest), the random nature of the size and concentration of the gas bubbles at the point of measurement in the stirred tank results in ‘noise’, which is shown as data scatter in the cell density measurements. For certain applications, such as fluorescence tagging, this is a problem as in such cases it is necessary to detect very small changes in absorbance, at values beyond the standard level of precision. In addition, the rate of aeration and agitation may vary during fermentation, such that the effect of bubbles on the measurements made is also variable.
Although other methods may be used to monitor the growth of microorganisms during aerobic fermentation (such as measurement of carbon dioxide production rate, oxygen consumption rate or fluorescence), the preferred method involves measurement of the cell density of a culture because it is less sensitive to culture conditions, such as temperature and pH. In most cases, the measurement of the culture's optical density is carried out ‘off-line’ using a spectrophotometer, typically in the region of 600 nm. By way of explanation, ‘off-line’ measurement of the optical density of a culture involves removing a sample of medium and measuring the optical density of the sample using an external spectrophotometer. However, such ‘off-line’ measurement typically has the disadvantages that it may increase the time, cost, and loss of culture volume and also increases the risk of contamination of the fermenter. In addition, using conventional methods, the cell density of a culture may only be directly measured accurately for values below 0.8, preferably below 0.7 optical density (OD) units. This is due to the fact that above these values the relationship between the cell density and optical density is no longer linear. In order to measure OD values above 0.7-0.8, the samples must be diluted and the resulting measurements multiplied by a dilution factor, such that the OD value does not exceed 0.8 units and preferably does not exceed 0.7 units. However, such dilution may introduce errors in the determination of cell density and renders on-line measurements of OD impossible.
By way of explanation, the ‘on-line’ measurement of the optical density of a culture involves the use of an in situ probe immersed in a culture medium to monitor changes in the cell density of a culture during fermentation. However, such methods have the added complication that air bubbles must be removed from the culture medium prior to measurement, in order to produce an accurate and reliable determination of the optical density. Thus, such methods typically require the addition of a separate device to degas the medium and consequently do not provide a method for a continuous measurement of the optical density of the medium.
The use of an on-line probe for monitoring concentration changes in a culture is known in the art. Shiloach and Bahar (Sixth European Congress on Biotechnology, Florence, Italy, 1993) disclose the use of a sterilisable, on-line sensor (Cerex Corporation) that is capable of following changes in culture turbidity. The probe is based on light emission from a culture and provides a good correlation to off-line turbidity measurements. The probe can be interfaced to a data acquisition and control system such that it can provide direct measurements during the fermentation process. During operation of the probe, a teflon plunger with an embedded magnet moves up and down as a result of an alternating magnetic field. The plunger movement allows culture medium to flow into a sampling chamber provided within the probe and facilitates removal of air bubbles from the culture medium. Typically, the plunger movement cycles such that it is open for one minute and closed for one minute providing a new optical density value every two minutes. Thus, although the on-line sensor overcomes the requirement associated with off-line systems wherein a sample of culture medium is removed from the fermenter in order to measure the optical density externally using a spectrophotometer, the probe disclosed by Shiloach and Bahar does not allow continuous measurement of the optical density.
Combs and Bishop (Annual Meeting of the Society for Industrial Microbiology, Toronto, Canada, 1993) disclose the use of a similar on-line probe (Cerex Corporation) to measure the optical density of a culture during fermentation. During operation of the probe, a solenoid opens, culture medium enters the sampling chamber, a valve closes to de-bubble the culture through a riser port and the optical density of the sample is measured. Thus, the operation of the probe requires the opening and closing of a valve, such that the on-line measurement of the optical density of the medium is not continuous.